1. Field of the Invention
This invention relates to apparatus and the method for preparing corneal tissue for surgical graft, implantation or transplantation referred to herein as "surgical techniques" or "surgical procedures." Use of the apparatus and method for preparing the corneal tissue avoids significant modification of the ultrastructure of corneal tissue during preparation of the tissue samples themselves.
Although the phrases "corneal tissue" and "corneal tissue samples" are used throughout this disclosure it should be understood that the terms are intended to include reference to corneal tissue as well as other ocular tissue that may be suitable for grafting, implantation or transplantation. Current medical technology has verified that corneal tissue is ideal tissue for surgical use. It is possible now to even cryo-lathe corneal tissue to achieve the desired parameters for visual correction in the patient.
The contemplated utility of the apparatus of this invention is not limited to specific types or sizes of tissue, rather it should be understood to refer to any tissue made up from cells. The apparatus of this invention can be designed or adapted to any size, shape or type of cellular tissue. Therefore, the terms "tissue" and "tissue samples" are used interchangeably and are not limiting on the uses to which the method and apparatus of this invention can be placed. It should further be understood that when the terms "tissue" or "tissue samples" are used the reference is to corneal tissue.
Historically, it has been very difficult to retain donor corneas for use in future surgical techniques because of the inherent loss of viability and contamination that occurs during the conventional tissue maintenance, retention and preparation processes. It is thus essential that these be avoided wherever possible.
The "preparation" of corneal tissue should be understood to refer to preparation of tissue for analysis as well as the cryofixation of tissue in anticipation of grafting, implantation, transplantation, or modification. The apparatus of this invention can be used to prepare tissue for any medical or analytical procedure without the ultrastructural damage previously thought to be inevitable in cryopreparation.
The apparatus of this invention is to be distinguished from contemporary freeze-drying apparatus. Freeze-drying is a technique which is well known in the art together with the equipment necessary to implement such freeze drying. See, for example, U.S. Pat. No. 4,232,453. Freeze-drying normally contemplates sample temperatures of -50.degree. C. to -80.degree. C. In contrast, the cryopreparation of this invention contemplates sample temperatures of -120.degree. C. or below.
Conventional technology for maintaining and preserving corneal tissue prior to transplantation is by use of what is referred to in the technology as the McCarey-Kaufman media. The McCarey-Kaufman tissue media typically will maintain the viability of corneal tissue for approximately 4 to 5 days. The primary advantages of the McCarey-Kaufman media technique are its simplicity and expense as well as the ability to transport the tissue sample from one area to another. However, the obvious drawback is the short term (4 to 5 days) preservation of the corneal tissue.
The critical need to expand the 4 to 5 day preservation period to a substantially longer timeframe has resulted in some corneal surgeons using cryopreparation techniques for maintaining corneal tissue in a viable state prior to its use in transplantation surgery or other surgical procedures. Existing cryopreparation technology, much of which has been described hereinabove and will be described hereinbelow, includes a slowly controlled freezing of tissue and maintenance of the tissue in liquid nitrogen. These conventional cryopreparation techniques offer an advantage of long term storage but the critical disadvantage of inherent damage to the corneal tissue. The corneal tissue is damaged by ice crystal formation during both the freezing the thawing process. It is also inconvenient and difficult to transfer the tissue in a liquid nitrogen media.
The cryopreparation technique which is the subject of this disclosure offers the distinct advantage of long term preservation and at the same time eliminates the disadvantages of tissue deterioration and destruction due to ice crystallization and transportation.
As disclosed hereinbefore, the formation of ice crystals and artifacts during freezing is kept to a minimum by the cryopreparation of the tissue and cryofixation in less than one second. No other available methodology for cryopreparation of corneal tissue involves the extremes of temperature and pressure and concomitant rapid heat transfer which are present in the process and apparatus of this invention. The term "artifact" refers to a product of artificial character due to extraneous agency. The net result of ultrastructural translation or damage to corneal tissue is that the tissue is not suitable for use in surgical techniques.
The corneal tissue is rapidly supercooled with a "bounce free" apparatus with the endothelial surface of the tissue facing the chilled metal block. Such a configuration of the corneal tissue during its initial vitrification insures that the tissue water within the corneal endothelium reaches the vitreous phase and that minimal ice crystal formation occurs in the corneal endothelium. The tissue is then dehydrated by the process of stimulated molecular distillation. Since all water has been removed from the corneal tissue before it is allowed to devitrify there is no danger of ice crystal formation with resultant ultrastructural damage during the thawing process as is the case in freeze drying techniques. In addition, once the tissue has been dried it can be maintained indefinitely under low vacuum or in a dry inert gas atmosphere. It is also feasible to transport the dried and cryoprepared corneal tissue from one area to another, typically under conditions of low vacuum or dry inert gas.
When the corneal tissue is being prepared for transplantation, the vacuum or dry inert gas is removed and the corneal tissue is rehydrated with a balanced salt solution for an effective time period, typically 15 to 20 minutes. The corneal tissue is then ready for use in surgical techniques, such as transplantation, according to the standard surgical procedures for corneal transplantation. Recent information on such surgical procedures is provided by textbooks such as Smolin, G. and Throft, R., The Cornea, Scientific Foundations and Clinical Practice, 437-464 (1983) and Casey, T. and Mayer, D., Corneal Grafting, Principles and Practice, 81-86 and 271-280 (1984).
In addition to preserving a full thickness cornea, the process of this invention as well as the apparatus used in the process of this invention can also be used to preserve partial thickness corneal tissue. Partial thickness corneal transplants are commonly referred to as lamellar transplants. Recently a great deal of interest has evolved concerning the use of lamellar corneal tissue (partial thickness corneal tissue) for the correction of myopia (nearsightedness), hyperopia (farsightedness) and keratoconus. Current techniques employed to obtain corneal tissue used for lamellar transplants involve the use of relatively slow freezing (10 to 30 seconds) using a carbon dioxide jet with concomitant formation of deleterious ice crystals within the cellular architecture.
By using the ultra-rapid cryopreparation technique of this invention the vitreous state of water is achieved within the substructure of the corneal tissue and thereby the ultrastructural damage and inherent artifact formation are avoided which are inherent in previous freeze drying techniques.
The extreme low temperatures and vacuums used in the practice of the apparatus of this invention have generated unique problems not associated with freeze-drying apparatus. For example, sealing devices such as squeezable O-rings made from elastomeric material do not function effectively at these anticipated cryopreparation temperatures and vacuums. Therefore, it is necessary that cryopreparation apparatus be designed to seal various structures at the extremes of temperature and pressure encountered, i.e., the sample chamber to the rest of the apparatus. This is but one example of problems which have been encountered in the design of and which are unique to cryopreparation apparatus. For purposes of this application the terms "cryopreparation" and "cryofixation" are used in distinction to conventional "freeze drying" technology (-50.degree. to -80.degree. C.).
The vacuum levels disclosed and used in the apparatus of this invention cannot be achieved safely with prior art freeze drying equipment. Typical of previous methods for drawing vacuums in freeze drying methods and apparatus is the above-mentioned U.S. Pat. No. 4,232,453 which discloses the use of molecular sieves in glass containers. Molecular sieves in easily compromised containers cannot be used safely to create and maintain the required vacuum levels to achieve the partial pressures required for sublimation of water at the anticipated temperatures (-120.degree. C. or below) created by the apparatus of the disclosed invention.
2. The Prior Art
Inherent in prior art processes for corneal tissue preparation is the concomitant damage to and deleterious modification of viable tissue characteristics. These tissue characteristic modifications represent a severe limitation on their utility for corneal surgery.
Similarly, it is essential that cryopreparation methods and apparatus develop concurrently with other medical technology, i.e., surgical transplant techniques, bio-engineering and biogenetics. In short, cryopreparation is an essential intermediate step in evolving processes using or analyzing cells or tissue. If cryopreparation apparatus does not evolve then the thrust of medical technology into unexplained and unexplored medical arts will be blunted. The apparatus of this invention represents the cryopreparation breakthrough that will permit research into the use and preparation of biological tissue to keep pace with other advances in medical technology.
The most common method for preparing corneal tissue is freeze drying cryofixed tissue. Freeze-drying following cryofixation is a well documented and well known technique for tissue preservation. It has several advantages. Freeze-drying results in a near-instantaneous arrest of cellular metabolism. There is also a stabilization and retention of soluble cell constituents through elimination of solvent contact with the sample. These are significant advantages to freeze-drying that have resulted in a great deal of research in attempting to apply cryofixation and freeze-drying techniques to known tissue preparation processes.
Unfortunately, freeze-drying technology inherently possesses a number of disadvantages relevant to tissue preparation methodologies. The primary disadvantage in currently available freeze-drying techniques and apparatus is the inherent formation of ice crystals. As can be readily appreciated, the formation of ice crystals destroys the ultrastructural integrity of the tissue sample being reviewed. The infrastructure is distorted and the cytoplasm becomes reticulated. The formation of ice crystals in the sample can also result in a change in pH within microcompartments of the tissue (eutectic formation) which possibly can result in abnormal tertiary conformation of macromolecules. There is also the possibility that proteins will denature and precipitate. These are but a few of the disadvantages which are inherent in the freeze-drying process.
This general topic is discussed in some detail together with other prior art methods in an article entitled Freezing and Drying of Biological Tissues for Electron Microscopy, Louis Terracio and Karl G. Schwabe, published in The Journal of Histochemistry and Cytochemistry, Volume 29, No. 9 at pp. 1021-1028 (1981). Problems associated with artifact formation are described in Understanding the Artefact Problem in Freeze-Fracture Replication: A Review, The Royal Microscopial Society, (1982) at pp. 103-123.
The application of cryogenics to surgical procedures and specifically ocular surgical procedures is not entirely new. See for example U.S. Pat. No. 3,942,519 which discloses ultrasonic and cryogenic instrumentation adapted for the removal of cataracts in surgical operations. In addition, see U.S. Pat. No. 4,056,855 which discloses a method for implanting an intraocular lens. The patent mentions the historical use of cryogenic probes to remove cataracts. Reference is also made to U.S. Pat. No. 4,336,691 disclosing a "Cryojet Rapid Freezing Apparatus" which can be used to rapidly freeze specimens such as corneal endothelium. The freezing in U.S. Pat. No. 4,336,691 is typically for the purpose of electron microscope examination and not transplantation.
A general principle found applicable to freezing techniques, which has demonstrated utility in the preparation of tissue samples, is that as the cooling rate increases, tissue fluids can be vitrified without the separation of water to extracellular spaces. It has been postulated, that regardless of the rate of cooling, ice crystals may still be formed, but as the cooling rates increase the size of the intracellular ice crystals decreases. The small size or absence of ice crystals at high freeze rates is of course a substantial advantage in morphology retention as this results in minimal artifact creation and minimal ultrastructural damage during tissue dehydration. The apparatus of this invention requires the rapid supercooling of tissue samples to the vitreous phase in less than one second followed by dehydration of the tissue sample while in the state of reduced partial pressure of water vapor, all without substantial ultrastructural damage to the tissue cells.
Historically, the criteria by which the techniques for rapid supercooling have been judged was not the cooling rate of the system but simply the temperature of the environment in which the tissue was frozen. Thus, the term rapid supercooling has been applied to any system in which the supercooling agent has a temperature of -150.degree. C. or below. The effectiveness of a cooling system is dependent upon the rate at which heat is removed from the sample. Heat transfer is dependent not only on the temperature of the freezing system but also on its physical and thermal characteristics, as well as the size and thermal characteristics of the tissue.
The most commonly used technique for rapid supercooling is to immerse or "quench" the sample in a fluid cooling bath. The most commonly used fluids for quenching are liquid nitrogen, isopentane, propane and fluorocarbons such as Freon 12 and Freon 22. Although liquid nitrogen is generally regarded as an ideal quenching fluid due to its low temperature (-196.degree. C.), there are inherent disadvantages in the use of liquid nitrogen due to the occurrence of tissue surface film boiling caused at least in part by the low heat of vaporization of liquid nitrogen. Film boiling is a characteristic of liquid nitrogen that inhibits the heat transfer rates by actually insulating the sample.
An alternate prior method for rapid supercooling is freezing on the polished surface of a chilled metal block. This typically involves opposing the tissue sample to a polished flat metal surface by pressing it firmly against the surface of the metal. Silver and copper are typically used as the polished metal blocks. This method is designed to take advantage of the high thermal conductivities and heat capacities of these metals when cooled to liquid nitrogen or liquid helium temperatures. The critical step in chilling on the surface of a metal is making firm contact with the dry, chilled metal surface with no rotational, translational or rebounding motion. Certain commercially available apparatus having known utility in the medical arts address and provide "bounce-free" freezing. Credit for the development of this apparatus is generally accorded to Dr. Alan Boyne of the University of Maryland School of Medicine.
There has recently been verification that there is a direct correlation between cooling rate and ultrastructural preservation in quenching fluids. As the freezing rate increases over the range of 100.degree. C. to 4100.degree. C. per second (liquid nitrogen--propane), there is a corresponding decrease in the size of ice crystals formed and thus an improvement in morphological preservation. Use of such quenching fluids or other supercooling apparatus to vitrify a tissue sample in less than 1 second is preferred.
The critical steps in the subsequent tissue preparation process are invariably stimulated sublimation--dehydration of the supercooled tissue fluids, which have recently been described as a stimulated "molecular distillation" process. Once the appropriate supercooling method has been chosen and implemented, it is sometimes necessary to further process the corneal tissue.
For penetrating or full thickness transplantation the corneal tissue can be dehydrated with the apparatus of this invention and then stored or maintained for subsequent rehydration and transplantation. When refractive lamellar transplantation methodologies are indicated (epikeratophakia or keratomileusis) the cryoprepared corneal tissue may be modified by cryo-lathing the tissue so that its radius of curvature corresponds to an optical power appropriate to achieve visual rehabilitation. After these modifications are made it is then necessary to dehydrate the tissue with no concomitant ice crystal formation.
Thus, dehydration is an essential step in the preparation of biological tissue samples for storage. This step oftentimes results in tissue destruction via reticulation of the infrastructure if not performed under cryogenic conditions as disclosed. Tissue cell destruction from dehydration adversely affects the functional characteristics and viability of tissue masses being used, i.e. transplanted.
In certain prior drying techniques, the tissue sample had not been entirely solidified due to eutectic formation as the cellular fluid solutes were concentrated in bound water compartments. This transfer of solute occurs while the materials are in the fluid state when slow cooling is employed. When rapid cooling techniques are used, unique procedures which are distinct from those characteristic of freeze-drying, must be employed in the dehydration step. Problems result from the fact that dehydration must take place (the water must be removed) in the solid rather than the liquid state, i.e., via sublimation. An alternate procedure which has been used successfully is stimulated molecular distillation. Stimulated molecular distillation refers to a process in which the amount of energy in the antibonding orbitals of surface molecules is elevated, enabling them to escape to the gas phase and not be recaptured by the solid phase.
In the prior art, the freeze substitution approach has involved the removal of tissue water by substituting a solvent or solvent-fixative mixture for the solid phase water at -50.degree. to -80.degree. C. This introduces less severe solvent phase separation and chemical alteration artifacts to a tissue sample than past routine chemical fixation methodologies. From a practical standpoint freeze-drying is complicated by the requirement that the tissue sample be warmed to increase the vapor pressure of the supercooled water and allow sublimation to proceed in a reasonable period of time. The increased temperature, in addition to increasing vapor pressure can produce a series of physical events leading to the expansion of ice crystals and concomitant damage to the ultrastructural morphology of the tissue sample. Many of the physical events which occur during the warming process have to do with transitions in the physical state of the water which is present. Changes which are typically encountered are glass transition, devitrification and recrystallization with an ensuing series of crystal lattice configuration transitions.
Thus it can be appreciated that freeze-drying technology and cryopreparation techniques present an exceptional opportunity for the preparation of tissue samples for surgical techniques, i.e. grafting, implantation or transplantation. However, inherent in the use of freeze-drying techniques are problems associated with dehydration of samples. These are the problems which are addressed by the process and apparatus of this invention.
The cryopreparation process of this invention has demonstrated an extraordinary application in the transplanting of corneal tissue. Prior to this invention attempts to transplant corneas which involved a necessary freezing or freeze-drying of the corneas after removal from the donor invariably resulted in a clouded cornea upon transplanting. This physical condition of the transplanted cornea was caused by crystal formation in the cornea itself and concomitant damage to the stroma. Use of the apparatus of this invention has enabled ophthalmologists to cryoprepare corneas and to then transplant those corneas to recipients with virtually negligible clouding or crystal formation. The ability to so transplant corneas represents an exceptional advantage to the process of this invention as well as a medical breakthrough in corneal transplant surgery.
One advantage of the apparatus of this invention is the ability to cryoprepare tissue without overt disruption or destruction of the morphological characteristics of the ultrastructure of tissue cells. The apparatus of this invention permits the cryopreparation of tissue by dehydrating tissue maintained in the solid, vitreous phase without creating unnecessary artifacts.